Isolation and characterization of an antimicrobial Bacillus subtilis strain O-741 against Vibrio parahaemolyticus

Vibrio parahaemolyticus is a marine bacterium that can infect and cause the death of aquatic organisms. V. parahaemolyticus can also cause human foodborne infection via contaminated seafood, with clinical syndromes which include diarrhea, abdominal cramps, nausea and so on. Since controlling V. parahaemolyticus is important for aquaculture and human health, various strategies have been explored. This study investigates the application of antagonistic microorganisms to inhibit the growth of V. parahaemolyticus. We screened aquaculture environment samples and identified a Bacillus subtilis strain O-741 with potent antimicrobial activities. This strain showed a broad spectrum of antagonistic activities against V. parahaemolyticus and other Vibrio species. Application of the O-741 bacterium significantly increased the survival of Artemia nauplii which were infected with V. parahaemolyticus. Furthermore, the cell-free supernatant (CFS) of O-741 bacterium exhibited inhibitory ability against V. parahaemolyticus, and its activity was stable to heat, acidity, UV, enzymes, and organic solvents. Next, the O-741 CFS was extracted by ethyl acetate, and analyzed by ultra-performance liquid chromatography-mass-mass spectrometry (UPLC-MS/MS), and the functional faction was identified as an amicoumacin A compound. The organic extracts of CFS containing amicoumacin A had bactericidal effects on V. parahaemolyticus, and the treated V. parahaemolyticus cells showed disruption of the cell membrane and formation of cell cavities. These findings indicate that B. subtilis strain O-741 can inhibit the V. parahaemolyticus in vitro and in vivo, and has potential for use as a biocontrol agent for preventing V. parahaemolyticus infection.


Introduction
Vibrio parahaemolyticus is a prevalent foodborne pathogen in Taiwan and is a cause of gastroenteritis in many Asian countries [1].It is a gram-negative and halophilic bacterium that is widely disseminated in estuarine, marine and coastal environments [2].By consumption of contaminated raw or undercooked seafood, V. parahaemolyticus can cause human infection.V. parahaemolyticus is currently classified into 13 O serotypes and 71 K serotypes [3].Since the occurrence of pandemic O3:K6 strains in 1996, V. parahaemolyticus has gained global significance [4].Typically, the clinical isolates of V. parahaemolyticus express thermostable direct hemolysin (TDH) and produce ß-hemolysis on Wagatsuma agar, which is known as the Kanagawa phenomenon (KP) positive [5].Some KP-negative V. parahaemolyticus isolates are hemolytic and contain TDH-related hemolysin (TRH).TDH and TRH are the main virulence factors of V. parahaemolyticus [6].
In addition, V. parahaemolyticus can survive in fish and shellfish aquaculture, and cause infections in some cultured shrimps [7].Acute hepatopancreatic necrosis disease (AHPND), caused by this bacterium, is a severe shrimp disease that can lead to mortality and substantial economic loss [8,9].Thus, the prevention of V. parahaemolyticus infection is beneficial to aquaculture.Among several approaches investigated so far, applications of antagonistic microorganisms, disinfectants, antibiotics, antimicrobial peptides, botanical extracts, or lytic bacteriophages have been evaluated for control of this pathogen [10][11][12][13][14][15].As antagonistic bacteria, Bacillus species are widely distributed in nature, including marine environments, and are safe for use as probiotics [16][17][18].Some Bacillus species can express active natural products and exhibit a wide spectrum of antimicrobial activities against pathogenic bacteria [19,20].Therefore, Bacillus species are regarded as appropriate biological control agent (BCA) candidates for treating bacterial infections [17].
Recently, the significance of pathogenic V. parahaemolyticus in aquaculture and human health has increased [21,22], and aquaculture samples are a prominent source of this pathogen [23].It is increasingly necessary to control the risk of V. parahaemolyticus in aquaculture and the use of antagonistic bacteria seems like a promising option.In this study, we screened and identified an antimicrobial Bacillus subtilis strain O-741, and characterized its activity, stability and targeted V. parahaemolyticus cell response.Furthermore, its active antimicrobial compound was identified and the application of this strain was evaluated in vivo using Artemia nauplii.The results indicated that O-741 bacterium may be useful as a biocontrol agent against V. parahaemolyticus.

Strains and culture conditions
The Vibrio spp.used in this study are listed in Table 1.V. chloreae strains were stored in Luria Bertani (LB) broth with 30% glycerol (v/v).Other Vibrio spp.were stored in the same broth with 3% NaCl (LB-3% NaCl), and these bacterial strains were stocked at -80˚C.The strains were recovered from frozen stocks and cultured in LB broth or LB-3% NaCl at 37˚C with shaking at 160 rpm for 16-18 hours.

Isolation and screening of antimicrobial bacteria
A total of 1,545 bacterial isolates were isolated from fish gills, fish intestines, oysters and clams, which were collected within the cold chain of a fish market.The samples were homogenized, suspended in phosphate buffered saline (PBS), streaked on Tryptic Soy Agar (TSA)-3% NaCl plates, and incubated at room temperature for 1-2 days.The isolated colonies were screened for antimicrobial activities by spot inoculation on bacterial lawn with indicator bacteria.The V. parahaemolyticus strains D/4, KX-V231, V. harveyi strain S14, and V. vulnificus strain B5 were used as indicators.

Identification of antimicrobial O-741 bacterium
The O-741 bacterium which was isolated from oyster was cultured in LB for 16-18 hours, and bacterial cells were harvested by centrifugation.The genomic DNA from the cell pellet was extracted using a commercial DNA extraction kit (Genomic DNA Mini Kit, Geneaid Biotech).The 16S rRNA, gyrA and rpoB genes of the genomic DNA were amplified by polymerase chain reactions (PCR) with the primers shown in Table 2 [24,25].After DNA sequencing, the nucleotide sequences of amplified fragments were applied to homology search using the BLAST software of the NCBI.The phylogenetic trees were built in MEGA6, using the neighbor-joining method [11,26,27].The bootstrap values were calculated based on 1000 computer-generated trees.

Evaluation of antimicrobial activities of O-741 bacterium
The O-741 bacterium was grown in 100 ml LB broth at 37˚C, 160 rpm for 24, 48, and 72 hours and the bacterial cultures were separated into three fractions, the untreated bacterial cultures, bacterial pellets resuspended in LB broth, and cell-free supernatants (CFS).The CFS was collected by centrifugation at 16,000 rpm for 1 min and filtered through 0.22 μm polyethersulfone membrane (Merck Millipore, Ireland).The antagonistic activities of the prepared fractions were tested by well diffusion assays [28].Aliquots of 30 μl of the prepared fractions were added into 6-mm wells on LB-3% NaCl agar with different bacterial lawns (Table 1), incubated at 37˚C for 24 hours, and the diameters of the inhibition zones were measured.

In vivo challenge with the Artemia nauplii model
Axenic Artemia nauplii were obtained by a decapsulation and hatching process.Two hundred milligrams of Artemia cysts (Ocean Star International, Snowville, UT) were hydrated in double-distilled water (ddH 2 O) for 1 hour.Then, the sterile cysts were prepared and decapsulated [29].Briefly, 850 μl of NaOH (32%) and 12 ml of NaOCl (50%) were added to the suspension of hydrated cysts to facilitate decapsulation.The process was stopped after 3 min by adding 12 ml of Na 2 S 2 O 3 (10 g/l).The decapsulated cysts were washed with autoclaved artificial seawater (ASW) (ISTA, Taiwan).For the experiments, the cysts were hatched for 24-28 hours at 25˚C on a shaker at 80 rpm.After 24-28 hours of hatching, batches of 25 Artemia nauplii were counted and transferred to 6 cm petri dishes containing 10 ml of autoclaved ASW.Finally, the dishes were returned to the incubator and kept at 25˚C [9,30].The 25 Artemia nauplii were collected and transferred to a 6 cm petri dish containing 10 ml ASW, and infected with different concentrations (2.5 × 10 9 , 5.0 × 10 9 , or 1.0 × 10 10 CFU) of V. parahaemolyticus strain KX-V231 or YAS1206-16.The control group of Artemia nauplii was not infected with V. parahaemolyticus.After incubation at 25˚C for 72 hours, the survival rates of Artemia nauplii were recorded [31].The experiments were conducted in triplicate.
To assay the protection of O-741 bacterium against V. parahaemolyticus using the Artemia nauplii model, groups of 25 Artemia nauplii were incubated with different concentrations (1 × 10 8 , or 1 × 10 9 CFU) of O-741 bacterium, then infected with 5 × 10 9 CFU of V. parahaemolyticus strain KX-V231 or YAS1206-16 in 10 ml ASW at 25˚C.After incubation for 72 hours, the survival rates of Artemia nauplii were recorded.The control group of Artemia nauplii was not incubated with O-741 bacterium and infected with V. parahaemolyticus strains.The experiments were conducted in triplicate.

Evaluation of the stability and antimicrobial activity of O-741 CFS
The antimicrobial activities of O-741 CFS, which had been subjected to different stress treatments, were determined by well diffusion assays against V. parahaemolyticus strains KX-V231 or D/4.
To determine thermal stability, the CFS was heated at 37˚C, 60˚C, 80˚C or 100˚C for 30 or 60 min.The CFS was also digested by lysozyme (Sigma-Aldrich, St. Louis, MA, USA), proteinase K (Sigma-Aldrich, St. Louis, MA, USA), pronase (Sigma-Aldrich, St. Louis, MA, USA), catalase (Sigma-Aldrich, St. Louis, MA, USA), pepsin (Merck, USA) and trypsin-EDTA (Sigma-Aldrich, St. Louis, MA, USA) at 0.5 mg/ml concentration, at 37˚C for 2 hours.The CFS was incubated at 37˚C for 2 hours with acetone, acetonitrile, ethanol, ethyl acetate, ethyl ether, and methanol at concentrations of 10%.The CFS was adjusted to different pH values of 2, 4, 6, 8, 10, and 12 by HCl or NaOH, and incubated at room temperature for 1 hour.The CFS was also subjected to UV irradiation by being placed 75 cm from a 30W UV light source for 1 to 5 hours.

Extraction and analysis of antimicrobial compounds
For extraction of antimicrobial compounds, the O-741 CFS was extracted with 1:1 (V:V) ethyl acetate by stirring for 2 hours at room temperature.The organic phase and aqueous phase were condensed with a refrigerated centrifugal concentrator, dried in a rotary evaporator, and dissolved with methanol or water, respectively.Then, the crude extracts were filtered through a 0.22 μm polyethersulfone membrane (Merck Millipore, Ireland).
To estimate the antimicrobial activities of crude extracts, disk diffusion assays were used.Briefly, 10 μl crude extracts from the organic layer or the aqueous layer were dropped onto a 6 mm paper disk on a LB-3% NaCl plate with a bacterial lawn.Then, the plates were incubated at 37˚C for 16-18 hours.The antimicrobial activities were measured as diameter (mm) of the inhibition zones.
The fractions were then subjected to UPLC-MS/MS analysis.An Agilent 1290 Infinity II ultra-performance liquid chromatography (UPLC) system (Agilent Technologies, Palo Alto, CA, USA) coupled online to the Dual AJS electrospray ionization (ESI) source of an Agilent 6545XT quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) was used in this experiment.The samples were separated using an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters Corp., Milford, MA, USA).The mobile phases were ddH 2 O (eluent A) and acetonitrile (eluent B), both eluents had 0.1% formic acid.The column temperature was 40˚C.The instrument was operated in positive fullscan mode.
The effective compounds were eluted according to the following linear gradient: first, starting at 80% eluent A and 20% eluent B, eluent A was linearly decreased to 0% with an increase of eluent B to 100% in 23 min and then maintained for 3 min at a flow rate of 0.3 ml/min.

Scanning electron microscopy of V. parahaemolyticus cells
The V. parahaemolyticus cells treated with organic extract of O-741 CFS at 0 μg/ml or 50 μg/ ml were examined by Scanning Electron Microscopy (SEM).In addition, the bacterial cells were treated with methanol as control.Then, the cells were harvested by centrifugation and fixed in 4% paraformaldehyde and 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated through ethanol solutions, and dried in CO 2 medium with a critical point dryer (Hitachi HCP-2).After coating with gold/palladium, observation was performed under a field emission scanning electron microscope (FE-SEM, Hitachi S-4700) [33].

Statistical analysis
Triplicate experiments were performed in this study, and the data about the bacterial growth experiments were measured in triplicate.The data were analyzed by using t-test at a significance level of α = 0.05, using SPSS for Windows version 11.0 (SPSS, Chicago, IL, USA).

Evaluation of the antimicrobial activities of O-741 bacterium
In order to determine the antimicrobial activities, the bacterial culture broths, bacterial suspensions, and cell-free supernatants (CFS) from O-741 bacterium (24-hour culture) were assayed against 5 clinical isolates and 10 environmental isolates of V. parahaemolyticus.The results showed that the O-741 bacterium had strong antimicrobial activity against V. parahaemolyticus.Further investigation of the antagonistic spectrum showed that O-741 bacterium had high antimicrobial activities against 8 isolates from different Vibrio species.These results indicate that O-741 bacterium exhibited broad-spectrum antimicrobial activity against Vibrio species (Fig 2).

In vivo challenge using an Artemia nauplii model
We used the Artemia nauplii model to investigate the in vivo infection of V. parahaemolyticus strains KX-V231 and YAS1206-16 in an aquatic environment.Seventy-two hours post-infection by these V. parahaemolyticus strains, survival of Artemia nauplii was markedly reduced thus demonstrating the virulence of these two V. parahaemolyticus strains in this model (S3 Fig).The inhibitory activity of the O-741 CFS from 24-hour culture was characterized with indicator V. parahaemolyticus strains KX-V231 and D/4.The results showed high stability against different environmental stresses (S1 Table ).The CFS was thermally stable.After being heated at 60˚C for 60 min, 84.16 or 82.26% of the inhibitory activity remained when assayed with V. parahaemolyticus strains KX-V231 or D/4, respectively.When the temperature was increased to 100˚C, more than 40% activity remained.The CFS was also resistant to digestion by different enzymes, since none of the tested enzymes (lysozyme, proteinase K, pronase, catalase, pepsin, and trypsin-EDTA) reduced the activities to lower than 90% relative to the untreated control.The activities of the CFS were stable to most of these organic solvents; acetone was the only solvent to decrease the activity to lower than 70%.In addition, the activities of the CFS were stable to a wide range of acidity treatments (pH 2 to pH 12), and UV irradiation.

Extraction and characterization of the O-741 CFS
For examination of the antimicrobial compound, the O-741 CFS from 24-hour culture was prepared, and extracted by ethyl acetate.After drying, the organic layer and aqueous layer were re-dissolved in methanol and ddH 2 O, respectively.Then, the crude extracts from the organic layer or the aqueous layer were assayed for antimicrobial activity.The results showed that the antimicrobial activity of crude extracts from the organic layer were markedly stronger than those from the aqueous layer, whereas 0.5 mg/ml crude extract from the aqueous layer showed 9.1-10.4mm inhibition zones in these two indicators, and 0.05 mg/ml crude extract from organic layer showed 14.7-15.6mm inhibition zones (Table 3).

Analysis of antimicrobial compounds from the organic extracts
The organic extracts from the 24, 48, and 72-hour O-741 cultures showed declined antimicrobial activity with prolonged culture time (Fig 4).In addition, analysis of organic extracts by UPLC-MS/MS spectrometry showed that the spectrum presented mass of peptides, and level of the dominant peak declined at 48, and 72 hours relative to 24 hours culture (Fig 5A).Furthermore, the fragmentation pattern at m/z 424 Da corresponded to amicoumacin A (Fig 5B ; Table 4) [11,33].These results indicated that O-741 bacterium can produce amicoumacin A and exhibit antimicrobial activities.The bacterial cultures of V. parahaemolyticus strains KX-V231, D/4, or YAS1206-16 were incubated with 50, 100, or 200 μg/ml of organic extracts from the 24-hour culture for 8 hours.Survival levels of these V. parahaemolyticus strains significantly decreased along with the increase in organic extract amount (Fig 6).These results demonstrated that the organic extract

Effect of antimicrobial compounds on cell morphology of V. parahaemolyticus
Following treatment with organic extract containing antimicrobial amicoumacin A, the morphology of V. parahaemolyticus cells was observed by SEM (Fig 7).The cells treated with organic extract at 0 μg/ml were rod-shaped (1,363 × 438 nm) with a smooth cell surface (Fig 7A and 7B).However, the cells treated with organic extract at 50 μg/ml were mostly coccoidshaped (693 × 670 nm) with irregular collapse that formed cavities in the cell surface (Fig 7C -7F).As a control, the cells were treated with methanol and maintained the rod-shaped morphology (Fig 7G and 7H).These observations indicate that the organic extract can disrupt the cell membrane and the cell wall of V. parahaemolyticus cells.

Discussion
Vibriosis is an illness caused by infection with Vibrio bacteria.Overuse of chemicals and antibiotics in controlling vibriosis can result in environmental pollution, and drug resistance issues [34].Drug-resistant microorganisms can greatly enhance the risk of infection, and have enormous impacts on aquaculture and human health [35].The use of biocontrol agents (BCA) is considered to be an environmentally-friendly approach to lower the threat of pathogenic Vibrio bacteria.
The Bacillus strains are appropriate biocontrol agent candidates for prevention of bacterial infections.Many Bacillus species have been proven to be safe, and some strains are used as probiotics for human and animal consumption [16].Recently, Bacillus species have been used to inhibit aquatic pathogenic bacteria, including V. parahaemolyticus [11,17], V. anguillarum [33], V. alginolyticus [36], V. cholerae [37], V. harveyi [38], and V. vulnificus [33].In this study, we screened and identified a Bacillus subtilis strain O-741 from an aquaculture environment.The O-741 bacterium and its CFS exhibited strong antimicrobial activities against 23 strains of 7 Vibrio species (Table 1).In addition, the O-741 bacterium was able to protect Artemia nauplii from infection with V. parahaemolyticus.The characteristics of the O-741 bacterium show a wide antagonistic spectrum and thus, this bacterium may be a good candidate for use in prevention of Vibrio bacterial infection.
Several pieces of research have reported the activity of the antimicrobial compounds in the CFS produced by B. amyloliquefaciens [39], B. pumilis [33,40] and B. subtilis strains [11,28] under physical and chemical treatments.The results of stability assays strengthen the evidence for the possible useful application of CFSs as fish feed additives [41].In this study, the stability and antimicrobial activity of the O-741 CFS were also investigated.The O-741 CFS was found to have inhibitory activity and be highly stable to heat, enzymes, organic solvents, pH, and UV treatments (S1 Table ).The results of the stability assays of O-741 CFS further support its possible efficient application under different environmental conditions.
In this study, the active antimicrobial compound in the O-741 CFS was identified to be amicoumacin A. Amicoumacin A was isolated for the first time from Bacillus pumilus by Itoh et al. [42].This compound has also been found in other Bacillus strains [11,33,[43][44][45].Amicoumacin A exhibits inhibitory activities against different bacterial pathogens, such as Helicobacter pylori, methicillin-resistant Staphylococcus aureus (MRSA) and Vibrio species [33,46,47].The anti-inflammatory and antitumor effects of amicoumacin A have also been described [42].We also showed that the amicoumacin A in O-741 CFS highly inhibited V. parahaemolyticus and some other pathogenic Vibrio bacteria that are responsible for Vibrio diseases in finfish, shellfish and shrimp [15,48].The broad antimicrobial spectrum of amicoumacin A may make it an effective prophylactic/therapeutic agent for Vibrio diseases in aquaculture.
Previous studies have indicated that amicoumacin A is the major metabolite accumulated at early incubation time points, but declines within 24 hours, and then appears as other amicoumacin derivatives [49,50].However, the derivatives of amicoumacin have weak antimicrobial activities [50].A similar accumulation, decline, and derivation of amicoumacin A was observed in O-741 CFS with changes in antimicrobial activity (Figs 4 and 5).The action of amicoumacin A has been reported in V. vulnificus, in which the cell was damaged by membrane poration [33].Furthermore, Lama et al. found that the amicoumacin A regulates the autolysis and activity of murein hydrolase in methicillin-resistant Staphylococcus aureus (MRSA) [47].The murein hydrolase is involved in turnover of peptidoglycan in the cell wall and daughter cell separation after cell division.Amicoumacin A exhibits antimicrobial activity through reduction of murein hydrolase activity.A recent study suggested that amicoumacin A can interfere with translation by locking the mRNA in the ribosome and be a protein synthesis inhibitor [51,52].These reports indicate that amicoumacin A uses multiple mechanisms to inhibit the bacteria.Our findings demonstrate that exposure to the organic extract containing amicoumacin A resulted in the transformation of V. parahaemolyticus cells from rod-shaped to coccoid-shaped form (Fig 7).The observations suggest that amicoumacin A has the capability to impact cell membranes, cell walls, and potentially affect the cellular processes responsible for cell morphology.It is worth investigating the detailed molecular mechanism(s) behind this structural alteration caused by amicoumacin A.
In aquaculture, V. parahaemolyticus is one of the major pathogenic Vibrio bacteria leading to high rates of mortality of aquatic organisms and massive economic losses [53].Artemia nauplii are aquatic invertebrates and the primary feed for farmed fish and shrimps [54].In addition, they have been used as a model for examination of V. parahaemolyticus infection [9,30].Our study demonstrated that Artemia nauplii is a valid model to assay the infection of V. parahaemolyticus strains, and in this model the O-741 bacterium provided significant protection against this pathogen (Fig 3).These results indicate that the O-741 bacterium may have potential applications in aquaculture.
In conclusion, a novel B. subtilis O-741 bacterium was isolated and its antimicrobial activities against various pathogenic Vibrio bacteria including V. parahaemolyticus, V. anguillarum, V. alginolyticus, V. chloreae, V. fluvialis, V. harveyi and V. vulnificus were evaluated.The functional compound of O-741 and its action on V. parahaemolyticus were identified, and its suitability for application in aquaculture was also verified by the Artemia nauplii model.Furthermore, our findings suggest that the antagonistic O-741 bacterium may be candidate for preventing Vibrio bacterial infection in humans, and may aid in reducing the potential risk of disease transmission.

For
selection of bacteria against Vibrio species, a total of 1,545 bacterial isolates were screened and characterized by spot inoculation against V. parahaemolyticus, V. harveyi, and V. vulnificus.The results revealed that the isolate O-741 bacterium had obvious antimicrobial activities, which was further confirmed by well diffusion assays against V. parahaemolyticus strains KX-V231, D/4 and YAS1206-16 (Fig1).For identification of O-741 bacterium, genome DNA was extracted and the 16S rRNA, gyrA, and rpoB genes were amplified by PCR (S1 Fig)and sequenced.The analysis of BLAST matching showed that the nucleotides sequences of 16S rRNA, gyrA, and rpoB genes had a high similarity to the Bacillus subtilis.The phylogenetic trees were constructed, and the results showed that the strain O-741 clustered with B. subtilis strains (S2 Fig).

Fig 1 .
Fig 1. Antimicrobial activities of isolated O-741 bacterium revealed by well diffusion assays.The O-741 bacterial culture broth, bacteria suspension and cell-free supernatant (CFS) were loaded into the wells of LB-3% NaCl plates inoculated with different strains of V. parahaemolyticus.Then, the plates were incubated at 37˚C for 24 hours.The results of well diffusion assays showed antimicrobial activities against V. parahaemolyticus strains KX-V231 (A), D/4 (B), or YAS1206-16 (C).The inhibition zones of O-741 bacterium against V. parahaemolyticus strains KX-V231 (D), D/4 (E), or YAS1206-16 (F) are shown.NC, LB broth was added as the negative control.Data shown are the mean in mm ± SE from three independent experiments.https://doi.org/10.1371/journal.pone.0299015.g001

Fig 2 .
Fig 2. Antagonistic activities of O-741 bacterium were evaluated by well diffusion assays.(A) The antagonistic activities of O-741 bacterium on 5 clinical isolates of V. parahaemolyticus.(B) The antagonistic activities of O-741 bacterium on 10 environmental isolates of V. parahaemolyticus.(C) The antagonistic activities of O-741 bacterium on 8 isolates of Vibrio spp.. Data shown are the mean in mm ± SE from three independent experiments.https://doi.org/10.1371/journal.pone.0299015.g002 After different culture times(24, 48, and  72 hours), the O-741 CFS were collected and the antimicrobial activities were examined by well diffusion assays.The O-741 CFS showed inhibitory activities against V. parahaemolyticus strains KX-V231, D/4, and YAS1206-16.The O-741 CFS from 24-hour culture exhibited the most significant inhibitory activities as compared to CFS from 48-or 72-hour culture (Fig 4).

Fig 5 .
Fig 5. Extraction and characterization of antimicrobial substances from organic extracts.The crude extracts from organic layer were analyzed by UPLC-MS/MS.(A) The spectrum shows the mass of peptides and the dominant peak (arrow).With prolonged culture time, the dominant peak declined.(B) The fragmentation pattern at m/z 424 Da corresponds to the compound amicoumacin A. https://doi.org/10.1371/journal.pone.0299015.g005